The development of new, broadly effective anti-HIV drugs that have little or no toxicity is a high-priority NIH goal for AIDS research. The HIV-1 integration reaction proceeds in two steps, both of which are carried out by integrase (IN). In the first step (3' processing or 3'P), IN removes a small number of nucleotides (usually 2) from the 3' ends of the linear viral DNA. In the second step, IN inserts the newly trimmed 3' ends of the viral DNA into host DNA; this reaction is called strand transfer, or ST. IN has only one active site that carries out both the 3'P and ST reactions. There are crystal structures of prototype foamy virus IN that show similarities and differences in the way that IN interacts with the viral DNA substrate in the 3'P and ST reactions. Because integration is an essential step in the virus life cycle, IN is an important target for antiretroviral drugs. The four approved anti-IN drugs (raltegravir, elvitegratir, dolutegravir, and bictegravir) all target the ST reaction, and are, for this reason, sometimes called INSTIs. Despite their relatively recent development, INSTIs are potent drugs with few side effects and are becoming increasingly important in antiretroviral therapy (ART). Cabotegravir has been formulated with rilpivirine and this combination has shown promise in long-term treatment strategies. However, as is the case with all anti-HIV drugs, INSTIs select for resistant strains of HIV. We are focusing on developing new anti-IN compounds that show little or no toxicity, and are broadly effective against the known drug-resistant mutants. We have made excellent progress in developing IN inhibitors that have low nanomolar potency in a one-round assay, retain potency against a broad panel of resistant mutants, and show little or no toxicity in cultured cells. Although we continue to make and test IN inhibitors, we have made sufficient progress that we have, with help from the NCI, begun to do pharmacokinetic testing on the best of our compounds. _____There are millions of retroviral integration sites in the host cell genome, but for many retroviruses, integration is far from random; we have been studying how HIV selects its integration sites. In the last few years, we have become increasingly interested in understanding what governs the distribution of HIV integration sites, both in cultured cells and in samples from patients. In the work that was done in cultured cells, we were part of a collaboration that investigated the ability of the host protein HRP2 to replace LEDGF in directing HIV integration to the bodies of highly expressed genes, and we showed that the host factor CPSF6 plays a key role in guiding the preintegration complex to regions of the genome that are gene rich and contain numerous highly expressed genes. LEDGF is a bipartite protein, in which the C-terminus binds IN and the N-terminus binds chromatin. We and others have used integration site analysis to show that there is extensive clonal expansion of HIV-infected cells in patients on ART. We showed in HIV-infected individuals on ART that more than 40% of the infected cells are in clones and that, in some cases, the integration sites can contribute to this expansion. We also showed that highly expanded clones can carry infectious proviruses, and release virions into the blood. Thus, clonal expansion of infected cells can contribute to the reservoir that has made it impossible to cure HIV infections with the currently available drugs. Although it has been proposed that viral replication plays an essential role in persistence and in the maintenance of the reservoir, even in fully compliant patients on successful ART, we think that the weight of the evidence supports the idea that there are patients in which ART completely blocks viral replication. Thus, it is important to understand the processes that allow infectious proviruses to persist even if viral replication is completely blocked. _____Given the importance of clonalexpansion in HIV persistence, we determined how soon after HIV acquisition infected clones can grow large enough to be detected (clones larger than ca.105 cells). We studied 12 individuals sampled in early HIV infection (Fiebig stage III-V/VI) and 5 who were chronically infected. The recently infected individuals were started on ART at or near the time of diagnosis. We isolated more than 6500 independent integration sites from peripheral blood mononuclear cells (PBMC) before ART was initiated and after 0.5-18 years of suppressive ART. Some clones of infected cells could be detected approximately 4 weeks after HIV infection and some of these clones persisted for years. The results help to explain how the reservoir is established early and persists for years. ____Our data provide a better understanding of the generation, maintenance, and persistence of the reservoir that has made it impossible to cure patients with the available anti-HIV drugs. However, there are limitations to the samples that can be obtained from HIV-infected patients. For that reason, we developed a simian immunodeficiency virus (SIV)/rhesus macaque model in collaboration with Dr. Jeffrey Lifson (Leidos Biomedical Research, Inc.) and showed that SIV-infected cells can clonally expand in infected macaques. The technology that we use to isolate large numbers of HIV integration sites was developed as part of the project in which we use redirected HIV integration to map the binding sites of host proteins, both WT and mutant, on chromatin. Although this HIT-Seq technology works well and has allowed us to answer some key questions about where and how important host factors bind to chromatin, we have redirected the personnel and resources that were dedicated to the HIT-Seq project to projects in which we are mapping HIV integration sites in patients and SIV integration sites in macaques. ______PATENTS LINKED TO THIS PROJECT: (1) Zhao XZ, Smith S, Metifiot M, Johnson B, Marchand C, Hughes SH, Pommier Y, Burke TR Jr: Compounds for Inhibiting Drug-Resistant Strains of HIV-1 Integrase. U.S. Patent #9,676,771 B2, issued June 13, 2017. (2) Zhao XZ, Smith S, Metifiot M, Johnson B, Marchand C, Hughes SH, Pommier Y, Burke TR Jr: Compounds for Inhibiting Drug-Resistant Strains of HIV-1 Integrase. U.S. Patent #10,208,305, issued February 19, 2019. (3) Burke TR, Hughes SH, Johnson B, Pommier Y, Smith SJ, Vu B, Zhao XZ (submitted in 2011): HIV Integrase Inhibitory Oxoisoindoline Sulfonamides. Patent pending: PCT/US2012/048169 (PC application). (4) Zhao XZ, Burke TR, Hughes SH, Johnson B, Marchand CR, Metifiot MA, Pommier Y, Smith SJ (submitted in 2013): Hydroxylamide-Containing Compounds with Improved Efficacy against Raltegravir-Resistant Strains of HIV-1 Integrase. Tracking number: E-093-2013/0-US-01 (US application).